Great strides have been made over the past 25 years in the functional imaging of the human body using positron emitters (e.g., carbon-11, fluorine-18, and iodine-124) and positron emission tomography (PET), and gamma-emitting isotopes (e.g. iodine-123) and single-photon emission computed tomography (SPECT). Arguably, the probe that has received the most attention is 2-deoxy-2-[18F]fluoro-D-glucose (2-FDG) and, indeed, this sustains the field of clinical PET. 2-FDG is the most widely used PET tracer in the world for in vivo assessment of regional glucose metabolic rates in humans. Approved diagnostic uses with PET include its use for determination of myocardial viability and detection of cancer, epilepsy, and Alzheimer's disease.
The success of 2-FDG PET imaging rests upon the finding that [14C]-2-deoxy-D-glucose can be used as a tracer to measure glucose metabolism in brain and other tissues. The human brain consumes about 125 grams of glucose per day and the body goes to extreme measures to deliver this amount across the blood brain barrier. Failure to supply the brain with glucose, for example in hypoglycemia, results in loss of consciousness and even death. Children with genetic defects in glucose transport across the blood brain have severe developmental problems (IQ, motor co-ordination, infantile seizures, etc).
Members of the GLUT family of genes are responsible for glucose uptake into brain across the blood brain barrier (GLUT1) and into neurons and glia (e.g. GLUT3). 2-FDG enters cells and crosses the blood-brain-barrier using facilitated glucose transporters (GLUTs). The glucose analog is phosphorylated by hexokinase to produce 2-deoxy-D-glucose-6-phosphate. Phosphorylated sugars are not substrates for the GLUTs, and 2-deoxy-D-glucose-6-phosphate is not further metabolized. Consequently, 2-deoxy-D-glucose-6-phosphate becomes trapped in cells. Similarly, the radiofluorinated 2-FDG is a substrate for GLUT transporters, is phosphorylated in cells to the 6-phosphate derivative, and becomes trapped.
The accumulation of 2-deoxy-2-[18F]fluoro-D-glucose-6 phosphate (2-FDG-6P) in cells permits, using Michaelis-Menten kinetics, determination of the local rates of glucose metabolism in all tissues. PET is employed to image 2-FDG-6P accumulation in living human subjects and animals. 2-FDG PET was first used to much advantage as an experimental tool to monitor regional brain activity in fully conscious subjects, and this revolutionized brain physiology. It was also found that 2-FDG-6P was accumulated in ischemic myocardium, and FDG PET has become a tool to study cardiac pathophysiology.
For at least fifteen years, 2-FDG PET has been used to detect tumors in the body (see, e.g., Czernin Acta Medica Austriaca 69: 162-170, 2002). This is based on the finding that most tumors have a high demand for energy in the form of glucose.
A second pathway for glucose entry into cells exists, the sodium/glucose cotransporter (SGLT) pathway (Wright & Turk (2004); Pflugers Arch. 447:510-518, 2004). However, 2-FDG is a very poor substrate for these glucose transporters, and so 2-FDG PET does not measure glucose utilization into cells by the SGLTs. A hydroxyl group in the equatorial plane of the pyranose ring at carbon-2 is required for binding and transport by SGLTs (Wright (2001) Am. J. Physiol: Renal Physiol. 280: F10-F18; Diez-Sampedro, et al. (2001) J. Biol. Chem. 276:52: 49188-49194). This means that mannose and 2-deoxy-D-glucose are poor substrates for SGLT1 and SGLT2. Mannose, however, is transported by SGLT4 (Tazawa, S. et al. Life Sciences 76: 1039-1050 (2005)).
Similarly, methyl-4-deoxy-D-glucopyranoside and methyl 4-fluoro-4-D-glucopyranoside (“Methyl-4-FDG” or “Me-4-FDG”) are not substrates for GLUTs (Wright et al (1980) Biochim. Biophys. Acta. 597:112 124 and unpublished observations), but are excellent substrates for SGLTs, including SGLT1, SGLT2, and SGLT4. The use of Me-4-FDG as an imaging probe for SGLT glucose transporters using PET or analogous imaging techniques (e.g., SPECT) has never been described.
There are two major members of the family of genes coding for SGLTs: SGLT1, which is thought to be expressed mainly in the small intestine, and SGLT2, which is thought to be expressed mainly in the kidney. SGLT1 is primarily responsible for the intestinal absorption of glucose and galactose in the human diet (180-200 grams per day), and mutations in the SGLT1 gene produce the disease Glucose-Galactose Malabsorption (Wright et al (2002) Cell Biochemistry and Biophysics 36; 115-121). SGLT2 is mainly responsible for the reabsorption of glucose from the glomerular filtrate in the kidney (180 grams/day), and mutations in this gene produce the condition known as renal glucosuria (Santer R, et al. J. Am. Soc Nephrol 14:2873-2882, 2003). SGLT4 is also expressed in the intestine and kidney where it appears to be involved in the absorption of D-mannose and fructose (Tazawa, S. et al. Life Sciences 76: 1039-1050, 2005).
It has been commonly believed that SGLT1, SGLT2, and SGLT4 were restricted mainly to the small intestine and kidney. However, it was recently discovered that these genes are expressed throughout the body, including in the heart, lung, brain, prostate, testes, and uterus (Wright & Turk (2004) Pflugers Arch. 447:510-518, Wright unpublished material; Zhou et al (2003) J. Cellular Biochemistry 90:339-346; Tazawa, S. et al. Life Sciences 76: 1039-1050, 2005), and even in metastatic lesions of some tumors (Ishikawa et al (2001) Jpn J. Cancer Res 92: 874-879). We have also found that glucose transporters belonging to the SGLT (SLC5) gene family are also expressed in the brain. SGLT2, SGLT4, and SGLT6 mRNAs are found at high levels in the human whole brain and cerebellum (Wright & Turk, 2004, Wright, unpublished observations) and immunocytochemical studies demonstrate that SGLT1 protein is expressed in specific regions of the hypothalamus, hippocampus, cerebellum and brain stem of rodent and human brains (Poppe, R., et al. (1997) J. Neurochemistry 69:84-94; Hirayama & Wright, unpublished observations).
Therefore, it is reasonable to postulate that the SGLTs play an important role in glucose metabolism in many organs in the body in health and disease. The SGLTs differ from the GLUTs in that they use the sodium gradient across the cell membrane to “pump” sugars into cells to a high concentration (e.g., against a concentration gradient). SGLT1 pumps a non-metabolized substrate (alpha-methyl-D-glucopyranoside) into cells to reach concentrations as high as 800-fold above plasma concentrations (Kimmich (1981) In: Physiology of the Gastrointestinal Tract, Edited by L. R. Johnson et al. Raven Press, New York, pages 1035-1061).
A [11C]-methyl-D-glucoside has been synthesized and biologically evaluated as a tracer of sodium dependent glucose transporters. (Bormans et al. J. Nucl. Med 44:1075-1081 (2003)). A commentary on this paper was published in the same issue of the journal. (Gatley. J. Nucl. Med. 44: 1082-1086 (2003)). In addition, three 18F-fluoro-n-alkyl glucosides have been synthesized and evaluated as potential substrates for sodium/glucose cotransporters. (De Groot et al. J Nucl Med. 44: 1973-81 (2003)). There are limitations with these probes in that the lifetime of [11C] is shorter than that of [18F] (20.4 vs. 109.8 minutes), and the affinity of the 11C- and 18F-fluoro-n-alkyl glucosides for SGLT1 is much lower than that for Methyl-4-[18F]-4-deoxy-D-glucose (0.8 to 2.6 mM vs. 50 uM).
Despite the advances made to date, a continuing need exists for efficient molecular imaging probes and methods for probing SGLTs and glucose metabolism in vitro and in living subjects, in healthy and diseased states. Particularly advantageous would be tracers and methods that enable researchers to assess the effect physiological role of the Na/sugar transporters in living human subjects, to monitor different therapeutic interventions on sodium-dependent glucose transport, in vitro and in vivo, and to probe and even distinguish between individual SGLTs (SGLT1, SGLT2, SGLT4, SGLT5, etc.). In addition, a need exists for SGLT probes that cross the blood-brain barrier and thereby enable SGLT activity in the brain to be monitored in vivo.